The present invention relates to receptors for growth factors, specifically to the fibroblast growth factor receptor (FGF-R). More particularly, it provides various purified fibroblast growth factor receptor proteins, nucleic acids encoding the receptor proteins, methods for the production of purified FGF-R proteins, proteins made by these methods, antibodies against these proteins, and diagnostic and therapeutic uses of these various reagents.
Polypeptide growth factors are mitogens that act on cells by specifically binding to receptors situated at the plasma membrane. These receptors usually have three major identifiable regions. The first is an extracellular region which contains the domain that binds the polypeptide growth factor (i.e. the ligand-binding domain). The second region is a transmembrane region and the third is an intracellular region. Many of these receptors contain a tyrosine kinase domain in the intracellular region.
The fibroblast growth factor receptor (FGF-R) proteins bind to a family of related growth factor ligands, the fibroblast growth factor (FGF) family. This family of growth factors are characterized by amino acid sequence homology, heparin-binding avidity, the ability to promote angiogenesis and mitogenic activity toward cells of epithelial, mesenchymal and neural origin.
The FGF family includes the following seven known FGFS:
(1, 2) acidic FGF (aFGF) and basic FGF (bFGF) (D. Gospodarowicz et al., Mol. Cell. Endocrinol., 46:107 (1986);
(3) the int-2 gene product (R. Moore et al., EMBO. J., 5:919 (1986);
(4) the hst gene product or Kaposi""s sarcoma FGF (K. J. Anderson et al. Nature, 332:360 (1988); M. Taira et al., Proc. Natl. Acad. Sci. USA, 84:2980 (1987));
(5) FGF-5 (X. Zhan et al., Mol. Cell. Biol., 8:3487 (1988)); and
(6) keratinocyte growth factor (J. S. Rubin et al., Proc. Natl. Acad. Sci. USA, 86:802 (1989)).
(7) FGF-6 (I. Marics, et al., Oncogene 3:335 (1989)).
The actions of acidic and basic FGF are mediated through binding to high affinity cell surface receptors of approximately 145 and 125 kDa (G. Neufeld and D. Gospodarowicz, J. Biol. Chem., 261:5631 (1986)).
The reference of Imamura et al., xe2x80x9cPurification of Basic FGF Receptors from Rat Brain,xe2x80x9d Biochem. Biophys. Res. Communications, 155:583 (Sep. 15, 1988) discloses the purification of nanogram amounts of a basic FGF receptor (bFGF-R) from rat brain.
While genes encoding a number of growth factor receptors have been molecularly cloned (e.g., mouse PDGF receptor, Yarden et al., Nature, 323:226 (1986), no clone has previously been identified as encoding a fibroblast growth factor receptor (FGF-R). Using antiphosphotyrosine antibodies to screen xcexgt11 cDNA expression libraries, a 2.5 kilobase cDNA encoding a novel tyrosine kinase gene, designated bek (bacterially expressed kinase), was isolated from a mouse liver cDNA library. (S. Kornbluth et al., xe2x80x9cNovel Tyrosine Kinase Identified by Phosphotyrosine Antibody Screening of cDNA Librariesxe2x80x9d, Mol. Cell. Biol. No. 8, 5541 (1988)). The bek sequence did not contain a transmembrane region and therefore could not be identified as a growth factor receptor. Another protein tyrosine kinase gene designated flg (fms-like-gene) was isolated from a human endothelial cell cDNA library by hybridization under relaxed stringency with a v-fms oncogene probe. (M. Ruta et al., xe2x80x9cA Novel Protein Tyrosine Kinase Gene Whose Expression is Modulated During Endothelial Cell Differentiationxe2x80x9d, Oncogene, 3:9 (1988)). Those authors could not identify a transmembrane region in their isolated sequence and therefore hypothesized that flg encodes a cytoplasmic tyrosine kinase.
The purified and cloned chicken bFGF and human bFGF receptors of this invention have amino acid sequence similarity with the bek and flg clones in the regions which have been isolated. However, both the bek and flg sequences reported were incomplete and there was no recognition of their function as FGF binding receptors. Moreover, the prior reports failed to recognize many of the structural and functional features described in the present invention.
Members of the FGF family appear to have roles in tissue development, tissue repair, maintenance of neurons and in the pathogenesis of disease. Aberrant expression of FGF may cause cell transformation by an autocrine mechanism. Moreover, FGFs may enhance tumor growth and invasiveness by stimulating blood vessel growth in the tumor or by inducing production of proteins such as plasminogen activator. However, identification of the components involved and understanding of the mechanisms and interactions involved remain woefully incomplete.
Purified FGF receptors and fragments, and isolated DNA sequences encoding defined FGF receptors and defined fragments (e.g., the ligand-binding domain) will greatly accelerate the understanding of fibroblast growth factor functions. Antibodies against specific and defined regions of the FGF receptor also become available. These reagents will find both diagnostic and therapeutic uses in the aforementioned processes. The present invention fulfills these and other needs.
The present invention provides purified fibroblast growth factor receptor (FGF-R) proteins, nucleic acids encoding FGF-R proteins, methods for the production of purified FGF-R proteins, purified proteins made by these methods, antibodies against these proteins and fragments, and diagnostic and therapeutic uses of these reagents. Notably, the present invention provides soluble and secreted forms of the receptors exhibiting an unusual receptor structure.
The present invention provides a method for modifying in vivo a fibroblast growth factor receptor modulated activity comprising administering to a patient an amount of a fibroblast growth factor receptor blocking agent effective to inhibit fibroblast growth factor binding to said fibroblast growth factor receptor. Typically, the agent will be a fragment of a human fibroblast growth factor receptor, e.g., a fragment produced in a cell transformed with a nucleic acid containing at least about 15 bases of a sequence selected from the group consisting of:
a) a DNA sequence in FIG. 3 or 4;
b) a sequence encoding a polypeptide of FIG. 3, 4 or 7; and
c) a sequence substantially homologous to a sequence of FIGS. 3 or 4.
The fragment will often be a fibroblast growth factor receptor extracellular domain without a tyrosine kinase region.
Alternatively, a method is provided for inhibiting binding between a fibroblast growth factor and a fibroblast growth factor receptor in a solution. This method will contain a step of combining an FGF-R peptide, e.g., a peptide homologous in sequence to a sequence described in FIG. 3, 4 or 7 to a solution or medium containing fibroblast growth factor and fibroblast growth factor receptor, usually native fibroblast growth factor receptor. Such methods will be useful in vitro, after employing labeled FGF-R peptide in assay procedures.
Compositions containing a soluble FGF-R polypeptide having between about five and two hundred contiguous amino acids from a human FGF-R extracellular domain are described. In one embodiment, the polypeptide contains at least about 80 amino acids from residues 1 to 287 of a human fibroblast growth factor receptor of FIG. 7 or an IgII or IgIII domain, or both. In alternative embodiments, the IgII domain will have about 7 contiguous amino acids from residues 85 to 141 of a human sequence of FIG. 7 or may contain a carboxy-terminal sequence substantially homologous to the 79 amino acid sequence from residues 222 to 300 of a soluble human protein of FIG. 7. Particularly preferred polypeptides consist essentially of the h4 or h5 sequences (FIG. 7).
A further aspect of the invention is a fibroblast growth factor receptor composition containing a substantially pure polypeptide of less than about 85 KDa comprising a fibroblast growth factor-binding domain. The polypeptide may be soluble or may specifically possess a signal segment, an IgI segment, an acidic segment, an IgII segment, an IgIII segment, an IgIIIT segment, or a transmembrane segment. Preferred embodiments will be homologous to a sequence described in FIG. 3, 4 or 7 or will include at least about 30 amino acids of each of both IgII and IgIII domains. The polypeptide can be one polypeptide chain in a multi-chain complex of proteins. A chicken fibroblast growth factor receptor is one preferred embodiment.
The present invention embraces isolated nucleic acids encoding human fibroblast growth factor receptor proteins which substantially lack an intracellular domain. Such a nucleic acid will usually exhibit a sequence homologous to an IgII domain described in FIG. 7, or may include a substantially full length IgII domain. The nucleic acid will usually also have a signal segment, an IgI segment, an acidic segment, an IgIII segment, an IgIIIT segment, a transmembrane segment, or a tyrosine kinase segment, and will preferably correspond to a sequence described in FIG. 3, 4 or 9. A particularly preferred embodiment is a nucleic acid encoding a receptor native to a human. The nucleic acids may be operably linked to a transcription promoter sequence and may further be incorporated into expression vectors suitable for production of recombinant FGF-R peptide.
Also included are isolated nucleic acids encoding a soluble human fibroblast growth factor receptor, preferably one homologous to h4 or h5. Protein products made by expressing such an isolated nucleic acid are provided.
A method is provided for making these proteins of newly recognized utility, e.g., fibroblast growth factor receptor activity, said method comprising expressing an isolated nucleic acid. Products produced by this method are now also available.
Additional methods are provided for making fibroblast growth factor receptor peptides by transforming a cell with a nucleic acid of at least about 21 bases of a sequence selected from the group consisting of:
a) a DNA sequence in FIG. 3, 4 or 9;
b) a sequence encoding a polypeptide of FIG. 3, 4 or 7; and
c) a sequence substantially homologous to a sequence of FIG. 3, 4 or 9.
Other methods for producing an antibody against a fibroblast growth factor receptor fragment are described, including a step of producing an antibody against a polypeptide epitope homologous to a sequence of at least six contiguous amino acids described in FIG. 3, 4 or 7. The epitopes of most interest will be those from a signal segment, an IgI segment, an acidic segment, an IgII segment, an IgIII segment, or an IgIIIT segment.
As a diagnostic use, these reagents provide a method for measuring a fibroblast growth factor or a fibroblast growth factor receptor in a target sample, said method comprising the steps of:
combining said target sample with a fibroblast growth factor receptor segment; and
determining the extent of binding between said segment and said sample.
This invention also provides a transformed cell capable of expressing a polypeptide homologous to at least a portion of a human fibroblast growth factor receptor. A preferred embodiment is where the cell expresses a polypeptide homologous to substantially the entire membrane bound or soluble form of a human fibroblast growth factor receptor.